Breast cancer is the most common type of cancer and the most common cause of cancer-related mortality among women worldwide. There is currently no cure for breast cancer and thus a greater understanding of the underlying biology of breast cancer will identify molecular targets and allow for the development of novel therapeutics. Cancer cells exhibit altered metabolism, characterized by increased glucose uptake and increased glycolysis under aerobic condition, a process known as Warburg effect. The exact molecular mechanisms underlying cancers dependency on metabolic pathways is unclear. Regulation of proteins by O- GlcNAcylation, post-translational modifications, is a reversible process that depends on glucose availability and is a powerful mechanism to regulate protein function. In this proposal, we will elucidate the mechanisms of how the nutrient sensor O-GlcNAc transferase (OGT), the enzyme responsible for catalyzing addition of O-GlcNAc to proteins, regulates cancer cell metabolism and survival stress signaling. Preliminary data suggests that O- GlcNAcylation regulates cancer metabolism via regulation of HIF-1 in a pVHL-dependent manner. Thus, we hypothesize that O-GlcNAcylation regulates cancer cell metabolism and survival stress signaling through VHL- dependent stabilization of HIF-1. In Aim #1, we will determine whether O-GlcNAcylation regulation of cancer cell survival requires ER stress and HIF-1 pathways. In Aim #2 we will determine how alterations in O- GlcNAc regulate the tumor suppressor VHL protein and determine its contribution to O-GlcNAc-mediated regulation on cancer cell survival metabolism and survival in vitro and in vivo. These studies will further our understanding of how metabolic reprogramming in cancer cells connects at the molecular level to survival stress pathways and identify novel anticancer pathways.